1. Field of the Invention
This invention relates to the field of oligonucleobases, such as oligonucleotides, for general use and possessing a new structural motif that enables the oligonucleobase to reversibly circularize.
2. Summary of the Related Art
Progress in the discovery and development of antisense oligonucleotides as therapeutic agents is continuing at a rapid pace (1-3). For the effective use of an oligonucleotide, it must interact with the target mRNA by Watson-Crick base pairing, activate RNase H for mRNA cleavage, be stable towards nucleases, and be taken up by cells efficiently (4,5). Oligodeoxynucleotide phosphorothioates (PS-oligonucleotides) possess all these properties and have been studied extensively for their in vitro and in vivo biological activity (6-10), safety (3,11-15), and pharmacokinetic profiles (15-19). The potential application of PS-oligonucleotides) as therapeutic agents is currently being evaluated in a number of human clinical trials (2). In order to further improve the potential of PS-oligonucleotides as antisense agents, we have introduced and evaluated various mixed-backbone oligonucleotides (MBOs) (20-24). In MBOs, the desirable properties of PS-oligonucleotides are maintained while undesirable properties are minimized by a combination of modifications in oligonucleotides. MBOs containing 2xe2x80x2-O-alkylribonucleotides have been studied extensively and have yielded promising results in terms of biological activity, in vivo stability, and general toxicity (21,25-27). Based on their advantages over PS-oligonucleotides, MBOs have become the first choice of second-generation antisense oligonucleotides and are currently being studied for their potential in human clinical trials.
In continuation of our efforts to improve the properties of PS-oligonucleotides as therapeutic agents, we have considered structural changes in PS-oligonucleotides. In our earlier studies we reported self-stabilized oligonucleotides, a PS-oligonucleotide containing a hairpin loop region at the 3xe2x80x2-end that provided increased in vivo nuclease stability and improved biological activity, and more importantly improvement in toxicity compared to a PS-oligonucleotide without a secondary structure at the 3xe2x80x2-end (12,28-30). Follow up studies by others have also yielded encouraging results (31).
In recent years, techniques based on the complementary hybridization between oligonucleotides and nucleic acid targets have also been widely applied in molecular diagnostics, therapeutics development, and mechanistic and molecular biological studies. As a result of human genome analysis, these techniques have become routine and there is an ever-increasing demand for more rapid, accurate, and effective nucleic acid detection and measurement methods. Fluorescence-based methods are more rapid and sensitive for hybridization detection and measurement than are the methods based on absorbance spectroscopy, calorimetry, and magnetic resonance spectroscopy. The advantage of the fluorescence-based techniques for monitoring complementary hybridization is that they can be used in both solution and solid-phase applications.
The polymerase-chain reaction (PCR) is extensively used in molecular biological and genetics based research and is increasingly becoming an essential tool for molecular diagnostics. Several homogenous fluorescence assay methods for probing amplification products in PCR reactions have been developed in recent years. These include TaqMan (40,41), molecular beacon (42), hairpin-primer (43), and scorpion (44).
Despite the advances that we and others have made, there is still a desire to develop antisense oligonucleotides having improved properties for use as therapeutic agents and in diagnostic applications.
The present invention provides a new structural class of oligonucleobases referred to herein as xe2x80x9cpseudo-cyclic oligonucleobasesxe2x80x9d (PCOs) or, equivalently, cyclicons. In PCOs, two oligonucleobases are linked to each other (directly or through a linker segment). One oligonucleobase, called herein the xe2x80x9cfunctional segment,xe2x80x9d (or, equivalently, the xe2x80x9cprimerxe2x80x9d or xe2x80x9cprimer-probexe2x80x9d segment) provides functionality to the PCO (e.g., the functional segment can be an antisense oligonucleotide), and the second, called herein the xe2x80x9cprotective segmentxe2x80x9d (or, equivalently, the xe2x80x9cmodifier segmentxe2x80x9d) is complementary to the terminal end of the functional segment (FIG. 1).
Under selected conditions, PCOs adopt an intramolecular cyclic or pseudo-cyclic structure as a result of complementarity between functional and protective segments, which form an intramolecular duplex (FIG. 1). Intramolecular duplex formation protects the functional segment, enhancing its stability. For example, when the functional segment is an antisense oligonucleotide and the functional segment and protective segment are connected via a 3xe2x80x2-3xe2x80x2-linkage, the linkage provides increased nuclease stability against 3xe2x80x2-exonucleolytic attack. The duplex formed between the antisense and protective segments provides additional nuclease stability against 5xe2x80x2-exonucleases.
These designer oligonucleobases may stay in linear form or hybridized form (FIG. 1) depending on the temperature, salt concentration, and length of the protective segment. If the PCO is in the intramolecular pseudo-cyclic form, it may exhibit fewer of the polyanionic-related side effects (e.g., complement activation and prolongation of partial thromboplastin time) known to occur with PS-oligonucleotides, because there are fewer exposed phosphorothioate linkages.
PCOs according to the invention can be made using standard techniques for synthesis of the constituent oligonucleobases and are useful for all purposes for which the functional oligonucleobase is useful.
The foregoing merely summarizes certain aspects of the invention and is not intended, nor should it be construed, as limiting the invention in any manner. All patents, patent applications, and other publications recited in this specification are hereby incorporated by reference in their entirety.